Salvelinus fontinalis (Brook Trout) are simultaneously the subject of eradication efforts in the western US and restoration efforts in the East. Thus, knowledge of their habitat requirements are important to management as well as ecological understanding of the species. Previous studies have evaluated habitat use and movement of established, resident Brook Trout, but none had looked at how transplanted Brook Trout respond in novel environments, nor has habitat selection been evaluated under different flow regimes that may detect differential use of primary habitat. We implanted wild Brook Trout with radio tags and tracked their movement for approximately 30 days during late spring 2002 and early spring 2003 in a central Appalachian stream. The hypotheses tested were: (1) there is no difference between habitat used by novel Brook Trout and available habitat, and (2) stream discharge levels have no effect on Brook Trout habitat selection. The daily tracking of fish in this study also permitted us to quantify fish movement. Brook Trout showed a preference for pool habitats—using them in greater proportion than availability—as well as a preference for large woody debris as cover. Overall, we found stream discharge did not affect habitat use. However, under low discharge levels, a negative relationship between discharge and pool use was detected, suggesting restriction to pool habitats under low flows. Home ranges of Brook Trout derived from radio telemetry averaged 450 m—similar to values obtained in other Appalachian studies employing mark-and-recapture methods. A comparison of our results with those of other studies suggests that Brook Trout released into novel environments move and select habitat similar to fish that have local knowledge of the environment.